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Biochimica et Biophysica Acta 1664 (2004) 189–197

The expression, activity and localisation of the secretory pathway

Ca2+-ATPase (SPCA1) in different mammalian tissues

Laura L. Wootton, Cymone C.H. Argent, Mark Wheatley, Francesco Michelangeli*

School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK

Received 27 January 2004; received in revised form 14 May 2004; accepted 28 May 2004

Available online 20 June 2004

Abstract

The distribution of the secretory pathway Ca2 +-ATPase (SPCA1) was investigated at both the mRNA and protein level in a variety of

tissues. The mRNA and the protein for SPCA1 were relatively abundant in rat brain, testis and testicular derived cells (myoid cells, germ

cells, primary Sertoli cells and TM4 cells; a mouse Sertoli cell line) and epididymal fat pads. Lower levels were found in aorta (rat and

porcine), heart, liver, lung and kidney.

SPCA activities from a number of tissues were measured and shown to be particularly high in brain, aorta, heart, fat pads and testis. As the

proportion of SPCA activity compared to total Ca2 + ATPase activity in brain, aorta, fat pads and testis were relatively high, this suggests that

SPCA1 plays a major role in Ca2 + storage within these tissues. The subcellular localisation of SPCA1 was shown to be predominantly

around the Golgi in both human aortic smooth muscle cells and TM4 cells.

D 2004 Elsevier B.V. All rights reserved.

Keywords: SPCA; SERCA; RT-PCR; Western blotting; Activity; Immunofluorescence

1. Introduction

Many hormones are known to mediate their action by

increasing the level of intracellular Ca2 +, via either the

opening of plasma membrane Ca2 + channels or by agonist-

induced production of inositol 1,4,5-trisphosphate (IP3) and

release of Ca2 + from intracellular stores via the IP3 receptor

[1]. Rises in intracellular Ca2 + in response to hormones are

known to regulate many processes including neurotransmis-

sion, fertilisation and transcription [2]. It is essential that this

rise in intracellular Ca2 + is transient and is brought back to

basal levels in order to prevent processes such as apoptosis

from occurring [3].

There are several mechanisms whereby intracellular

Ca2 + levels may be reduced, with the main focus being

on the plasma membrane Ca2 + ATPase (PMCA) [4] and the

sarco/endoplasmic reticulum (SERCA) Ca2 + ATPases [5].

More recently there has been interest in another intracel-

lular Ca2 + pump (pmr1), first isolated and cloned from the

0005-2736/$ - see front matter D 2004 Elsevier B.V. All rights reserved.

doi:10.1016/j.bbamem.2004.05.009

* Corresponding author. Tel.: +44-121-414-5398; fax: +44-121-414-

5925.

E-mail address: [email protected] (F. Michelangeli).

yeast Saccharomyces cerevisiae [6–8]. The mammalian

homologue of this protein is known as the secretory path-

way Ca2 + ATPase (SPCA), which, like SERCA and PMCA,

is a P-type transporting ATPase [11,18,19], and can trans-

port a single Ca2 + or Mn2 + for each ATP molecule hydro-

lysed. To date, two isoforms of the mammalian SPCA have

been identified (SPCA1 and SPCA2) which show 60%

sequence identity to each other [5].

In yeast and mammalian cells, pmr1 and SPCA have

been shown to be located mainly in the Golgi membranes

[5,10,11] where their function of transporting Ca2 + or Mn2 +

is believed to be important for the folding and glycosylation

of secretory proteins [9,12]. The Golgi apparatus has also

been shown to contain IP3 receptors on its surface [13],

suggesting that it is a possible agonist-releasable Ca2 + store

and thus the SPCA Ca2 + pump may also play a role in the

refilling of this store. Indeed, endogenous SPCA has been

shown to be responsible for baseline spiking in response to

histamine in HeLa cells [14] and in response to ATP when

overexpressed in COS-1 cells [15].

Two alternatively spliced forms of SPCA have been

identified in rat [16], each with molecular weights of

approximately 100 kDa. The two splice variants differ at

the carboxy-terminus, where one variant has an additional 4-

L.L. Wootton et al. / Biochimica et Biophysica Acta 1664 (2004) 189–197190

amino-acid extension as well as a substitution of val 919 for

phe [16]. Northern blot analysis of rat tissues has also shown

that SPCA is widely expressed, at least at the mRNA level.

The ATP2C1 gene encodes the human SPCA1 protein, of

which four splice variants have so far been identified [17].

These variants also have modifications at the carboxy-

terminus and vary in size from 888 to 949 amino acids

[17]. ATP2C1 is mutated in Hailey–Hailey disease (HHD)

[18,19], which is an autosomally dominant inherited skin

disorder, characterised by suprabasal cell separation of the

epidermis, known as ancantholysis.

In order to understand further the role and function of

this Ca2 + pump within different tissues, we have analysed

the expression of the SPCA1 protein in a variety of tissues at

the mRNA and protein level. In addition, we have also

measured its activity in these tissues as well as its subcel-

lular localisation in some cell types.

2. Materials and methods

2.1. Materials

Thapsigargin, uridine diphosphate (UDP), p-nitrophenyl

a-D-mannopyranoside and fluo-3 were all purchased from

Sigma. Thapsigargin and p-nitrophenyl a-D-mannopyrano-

side were both dissolved in DMSO, when used in experi-

ments the level of DMSO was never greater than 1% v/v.

Secondary antibodies conjugated to either horseradish per-

oxidase or fluorescein isothiocyanate (FITC) were purchased

from Sigma. The Y1F4 an anti-SERCA monoclonal anti-

body was a gift from Dr. J. M. East, University of South-

ampton. Wheat germ agglutinin conjugated with Texas Red-

X (WGATR) was purchased from Molecular Probes. All

other reagents used were of analytical grade.

In association with BioCarta GmbH, we raised and

purified a polyclonal antibody against the SPCA1 peptide

sequence LTQQQRDVYQQEKA in rabbit. This sequence

corresponds to amino acids 503 to 516 of the human SPCA1

sequence and from alignment studies with SERCA1 is likely

to correlate with a surface exposed region within the

nucleotide binding domain. As 13/14 of the amino acids

in this peptide sequence, to which the antibody was raised,

is identical for SPCA1 of all known sequenced mammalian

species (including rat and mouse), this antibody does

therefore cross react with SPCA1 in all animal tissues tested

in this study. It should also be noted that since the antibody

recognises an epitope in the middle of the protein it will

therefore recognise all splice variants forms of SPCA1, as

these differ only at the carboxy-terminus. Furthermore this

antibody is unlikely to cross-react with SPCA2, as the

sequence to which the antibody was raised showed only a

50% identity with SPCA1, with the differences uniformly

distributed throughout this sequence. This antibody was

specific for SPCA1, rather than other proteins, as pre-

incubation of the antibody with the peptide antigen sub-

stantially reduced its labelling of the SPCA1 protein band in

Western blots.

2.2. Reverse transcriptase polymerase chain reaction

(RT-PCR)

(a) Preparation of cDNA: mRNA was extracted from

various rat tissues, rat testicular derived cells and mouse

TM4 cells (a mouse Sertoli cell line), using QuickPrep

Micro mRNA purification kit from Amersham, follow-

ing the manufacturer’s instructions. The mRNA was

then converted to cDNA, using a First-Strand cDNA

synthesis kit (Amersham).

(b) Preparation of Testicular cells: The testicular cells

were prepared from Wistar rats by sequential enzymatic

digestion as descibed in Ref. [20]. Briefly, the testis

were aseptically removed, decapsulated and washed

three times in Dulbecoo’s PBS media supplemented

with 1 mg/ml D-glucose (DPSS+). Interstitial cells were

then lysed by incubating the tubules in DPSS+ (pH 7.2)

containing 1 mM glycine, 2 mM EDTA and 10 Ag/ml

deoxyribonuclease for 10 min at room temperature.

Lysed cells were removed by three washes in DPSS+.

The tubule fragments were then treated with 0.25% w/v

trypsin and 10 Ag/ml deoxyribonuclease at 32 jC for 35

min. Digestion was stopped by the addition of 0.1% w/v

soybean trypsin inhibitor and the tubule fragments

collected by centrifugation. These fragments were

washed three times in DPSS+ and then treated under

constant agitation with 1 mg/ml collagenase at 32 jCfor 30 min. Undigested material was allowed to

sediment under gravity for 30 min. The Sertoli/germ

cell fractions were collected by gentle centrifugation of

the post-collagenase fraction (60� g for 4 min); these

were grown in culture at 32 jC in 5% CO2/95% air in

DMEM/Ham’s F12 media (1:1 v/v) supplemented with

2% rat serum, 8 mM glutamine, 5 Ag/ml insulin, 5 Ag/ml transferrin, 100 U/ml penicillin and 100 U/ml

streptomycin. After 48 h in culture, the nonadherent

germ cells were removed by gentle washing of the

Sertoli cell monolayer with DMEM/Ham’s F12 (sup-

plemented as above) and collected by centrifugation

(400� g for 10 min). The Sertoli cells were recovered

by trypsinisation. The myoid cells were collected from

the supernatent of the post-collagenase low speed spin

step by centrifugation at 400� g for 10 min. Following

two washes in DPSS+, the myoid cells were plated in

DMEM/Ham’s F12 (1:1 v/v) supplemented with 10%

foetal calf serum, 8 mM glutamine, 100 U/ml penicillin

and 100 U/ml streptomycin. The Sertoli, germ and

myoid cells obtained by this method were judged to be

greater than 90% pure, assessed as described in Refs.

[46,47].

(c) PCR reaction: A 224-bp fragment of the rat SPCA1

cDNAwas amplified using Taq polymerase from 5 ng of

cDNA derived from various tissues/cells. The cDNAs

L.L. Wootton et al. / Biochimica et Biophysica Acta 1664 (2004) 189–197 191

used were determined to be devoid of genomic DNA

contamination. The forward primer used was 5V-AAACTGGAACCCTGACGAAG-3V and the reverse

primer used was 5V-GTGAGACTACCCTTTCGGTT-3V; these were as originally used in Ref. [21]. (These

primers are unique for SPCA1 and will not bind to the

cDNA of SPCA2). The PCR reaction condition was as

described in Ref. [21], with the exception of having 2

mM Mg2 + and 5% DMSO present. The PCR reaction

was then subjected to 35 cycles (which was shown to be

in the linear range when related to PCR product). Of

each total PCR reaction, 2.5 Al (10 Al of heart and aorta)

was resolved on a 2% w/v agarose gel in TBE buffer.

Each gel was stained with ethidium bromide before

visualisation using a UV transilluminator. Again it

should be noted that these primers are able to recognise

the cDNA from both splice variant forms of SPCA1 in

rat as well as mouse cDNA, due to identical nucleotide

sequences where the primers bind.

2.3. Preparation of rat microsomal membranes

Brain, lung, liver, testes, epididymal fat pads, heart,

spleen and kidney were obtained from male rats and aorta

was obtained from pig. Crude microsomal membranes were

prepared as described below. Each tissue was added to 5–10

volumes of ice-cold buffer (5 mM HEPES, 0.32 M sucrose,

0.1 mM benzamidine, 10 AM leupeptin and 0.1 mM PMSF,

pH 7.2). The tissues were chopped into small pieces before

being homogenised using a Polytron homogeniser. The

homogenate was centrifuged at 10,000� g, at 4 jC for 20

min. The supernatent obtained was further centrifuged at

100,000� g, at 4 jC for 1 h. The pellet obtained was

resuspended in buffer before being aliquoted and snap

frozen in liquid nitrogen prior to storage at � 70 jC.

3. SDS-PAGE gels and Western blotting

Microsomal membranes (15–30 Ag) were prepared in

sample buffer and heated at 60 jC for 15 min. The

membranes were then resolved on 7.5% (w/v) polyacryl-

amide gels, as in Ref. [22]. The proteins were transferred

onto nitrocellulose in transfer buffer (0.1% w/v SDS, 20%

v/v methanol, 25 mM Tris and 190 mM glycine), for 1 h at a

current of 750 mA, as in Ref. [23]. The nitrocellulose was

then blocked overnight in Tween 20–Tris buffered saline

(150 mM NaCl, 25 mM Tris–HCl, 0.05% v/v Tween 20, pH

8) (TTBS) with 5% w/v dried skimmed milk powder. The

blocking solution was removed and the nitrocellulose was

washed three times in TTBS. The nitrocellulose was incu-

bated with primary antibody diluted in TTBS with 1% w/v

BSA (1 in 100 dilution for anti-SPCA and 1 in 500 dilution

for Y1F4). The primary antibody solution was removed and

the blot was washed for 25 min with five changes of TTBS.

The nitrocellulose was then incubated for 1 h with second-

ary antibody conjugated with horseradish peroxidase in

TTBS according to the manufacturer’s instructions. The

nitrocellulose was then washed as before. The nitrocellulose

was incubated for 5 min with SuperSignal West Pico

Chemiluminescent substrate (Pierce) according to the man-

ufacturer’s instructions. The nitrocellulose was used to

expose Kodak BioMax film for varying lengths of time,

and the film was developed using a XO-graph developing

machine.

3.1. Quantification of Golgi enzyme markers in the

membranes

UDPase activity has been found to be associated with the

Golgi apparatus [24–26]. The UDPase activity of the tissue

membranes was assayed as follows: 40 mM HEPES pH 7.0,

10 mM CaCl2, 0.1% v/v Triton X-100, 2 mM UDP and 10–

20 Ag of membranes in a total volume of 0.5 ml were

incubated at 37 jC for 10 min. The reaction was stopped by

the addition of 0.25 ml of 6.5%w/v TCA, the tubes were

then chilled on ice for 10 min before being centrifuged at

low speed to remove denatured protein. To measure the

amount of phosphate liberated, 0.25 ml of each assay was

added to 1.5 ml of copper acetate buffer (11.25% v/v acetic

acid, 0.25% copper sulfate and 0.2 M sodium acetate, pH

4.0). Ammonium molybdate solution (0.25 ml; 5% v/v) was

then added to this and mixed. ELAN solution (0.25 ml; 2%

p-methyl-aminophenol sulfate containing 5% sodium sul-

fite) was added and mixed. The colour was allowed to

develop for 10 min before the absorbance was measured at

870 nm. Zero time incubations were carried out for each

tissue. A calibration curve was obtained by addition of

known amounts of phosphate to the reaction buffer, in the

absence of membranes (see Ref. [30]).

The enzyme a-mannosidase is also known to be associ-

ated with the Golgi apparatus [27–29]. The activity of this

enzyme was measured in a total volume of 1 ml of buffer

(40 mM HEPES, pH 7.0, 10 mM CaCl2, 0.1% Triton X-

100) and 10 mM p-nitrophenyl-a-D-mannoside with 5–20

Ag of microsomes. The accumulation of p-nitrophenol was

followed at 405 nm for 5 min at 25 jC.

3.2. Measurements of SPCA activity

Initial rates of ATP-driven Ca2 + uptake were measured

by monitoring the change in fluorescence of the calcium

sensitive dye fluo-3 [30–32]. Fluorescence changes were

monitored using a Perkin-Elmer LS50B spectrofluorimeter,

where excitation was set at 506 nm and emission was

measured at 526 nm. The fluorescence was related to the

[Ca2 +] using the following equation:

½Ca2þ� ¼ KdððF � FminÞ=ðF � FmaxÞÞ

where Kd is the dissociation constant for Ca2 + binding to

fluo-3 (900 nM at 37 jC, pH 7.2 in the presence of 100 mM


K+ [33]), F is the fluorescence intensity of the sample, Fmin

and Fmax are the fluorescence intensities in 1.25 mM EGTA

and 2 mM CaCl2, respectively. Membranes were added to a

stirred cuvette containing 2 ml of 40 mM Tris/phosphate,

100 mM KCl, 5 mM potassium oxalate, 2 mM sodium

azide, 2 AM vanadate, 1.25 AM fluo-3, 10 Ag/ml creatine

kinase and 10 mM phosphocreatine. The uptake of Ca2 +

was initiated by the addition of 1.5 mM Mg-ATP and uptake

rates were measured over a period of 300 s. To distinguish

between Ca2 + uptake caused by SERCA compared to that

caused by SPCA, experiments were repeated in the presence

and absence of 2 AM thapsigargin.

3.3. Tissue culture

TM4 cells, a mouse Sertoli cell line, were cultured at 37

jC, under 5% CO2/95% air, in DMEM/Ham’s F12 (1:1 v/v)

media (Gibco), supplemented with 5% v/v heat inactivated

foetal calf serum and 2.5% v/v horse serum. The human

primary aortic smooth muscle cells (HuASMC) used were

typically passaged between 12 and 14, and maintained at 37

jC under 5% CO2/95% air. The cells were grown in smooth

muscle cell basal medium supplemented with 12.5 Ag/ml

gentamycin, 12.5 ng/ml amphoteracin B, 5% foetal bovine

serum (FBS), 10 ng/ml epidermal growth factor, 3 ng/ml

basal fibroblast growth factor and 0.4 Ag/ml dexamethasone

(Promocell, Heidelberg, Germany). In order to convert the

cells into the contractile phenotype (which was the type

used in the study), the cells were then grown for 6 days in

the medium containing low FBS (0.25%) and in the absence

of epidermal growth factor, basic fibroblast growth factor

and dexamethasone [44].

3.4. Immunofluorescence

TM4 and human primary aortic smooth muscle cells

(HuASMC) were cultured on scratched glass coverslips and

gelatine-coated coverslips, respectively. The cells were

grown to approximately 80% confluence. The cells were

then washed three times in PBS. The TM4 cells were fixed

using 4% paraformaldehyde in PBS at room temperature for

15 min. The human primary aortic smooth muscle cells were

fixed at room temperature for 15 min with 2% formalde-

hyde. The coverslips were again washed three times in PBS.

The cells were permeabilised for 5 min at room temperature

with 0.1% v/v Triton X-100 in PBS. The permeabilisaton

media was removed and the coverslips were washed again

three times with PBS. The cells were incubated at room

temperature for 1 h with primary antibody. The primary

anti-SPCA1 antibody was diluted 1/100 in PBS and Y1F4

(anti-SERCA antibody) was also diluted 1/100 in PBS.

Following incubation with the primary antibody, the cover-

slips were washed three times with PBS. The coverslips

were then incubated with the appropriate secondary anti-

body conjugated with FITC. The coverslips were again

washed three times with PBS and once with distilled water.

The coverslips were dried of any excess liquid and were

mounted on glass coverslips using Hydramount (National

Diagnostics). The cells were visualised using a Leica

DMRB fluorescence microscope equipped with a Hama-

matsu ORCA camera.

3.5. Lectin affinity immunostaining

Wheat germ agglutinin (WGA) binds sialic acid and N-

acetyl glucosaminyl residues mostly found to be associated

with the Golgi complex [34]. TM4 cells were cultured on

scratched glass coverslips, fixed and permeabilised as de-

scribed above. The cells were incubated at room tempera-

ture for 1 h with WGA-Texas Red (WGATR) diluted 2 Ag/ml in PBS. The coverslips were then washed, mounted and

visualised as described above.

4. Results

Fig. 1A shows the results of RT-PCR on a variety of rat

tissues, except TM4 cells which are derived from mouse.

The primers were designed to amplify a 224-bp fragment of

the SPCA1 sequence from the cDNA of both rat and mouse.

The mRNA for SPCA1 was easily detected in testis, brain

(cerebrum) and cerebellar. Lower levels of detection were

observed in heart and aorta and levels were undetectable in

spleen and skeletal muscle. Fig. 1B shows the results of RT-

PCR from rat testis and testicular derived cell types. A 224-

bp fragment was detected in juvenile testis, adult testis,

Sertoli cells, TM4 cells, germ (spermatids) and myoid cells,

indicating the expression of SPCA1, at least at the mRNA

level, is abundant within testicular cells. A note of interest is

also that the level of SPCA1 mRNA expression appears to

be considerably less in juvenile testis compared to adult

testis suggesting that SPCA may be up-regulated during

development.

Fig. 2 shows the immunoblots of membranes derived

from a number of tissues and cells, probed with the anti-

SPCA1 antibody. These blots are from the same immunos-

taining procedure and are typical of three separate blots. The

detection of a band of f 100 kDa was observed in the

microsomal membranes from rat brain, rat lung, rat kidney,

rat heart, rat epididymal fat pads, rat testis, rat liver, porcine

aorta and mouse TM4 cells. The level of expression of SPCA

was particularly high in brain, testis, epididymal fat pads and

in TM4 cells. The expression was of a lower level in heart,

aorta, liver, lung, kidney, and not detectable in spleen. Fig. 3

shows the results of immunoblots of the same membranes

with Y1F4 (anti-SERCA antibody) [35]. The level of

SERCA is highest in heart, testis, lung and brain. Expression

is lower in epididymal fat pads, liver, spleen, aorta (pig) and

kidney. There is also a significant level of SERCA protein in

TM4 cells. A comparison between the levels of expression of

SERCA and SPCA in the different tissues shows that

although brain and testis appear to be abundant in both

Fig. 1. RT-PCR analysis of SPCA1 in rat tissue microsomes, pig aorta microsomes and the mouse TM4 Sertoli cells. Primers were designed, as originally

described in Ref. [21], to amplify over 35 cycles, in the presence of 2 mMMg2 + and 5% DMSO, a 224-bp sequence of the SPCA1 gene from 5 ng of cDNA. In

each of the lanes, 2.5 Al (10 Al for heart and aorta) of a 50-Al PCR reaction was resolved a 2% agarose gel, stained with ethidium bromide and visualised using a

UV transilluminator. (A) The lanes represent: (1) no cDNA, (2) rat testis, (3) rat heart, (4) rat aorta, (5) 100-bp ladder (NEB), (6) rat brain (cerebrum), (7) rat

cerebellar, (8) rat spleen and (9) rat skeletal muscle. (B) The lanes represent: (1) no cDNA, (2) rat juvenile testis, (3) rat adult testis, (4) rat Sertoli, (5) mouse

TM4, (6) 100-bp ladder (NEB), (7) rat germ cell and (8) rat myoid cell.


proteins, other tissues such as heart, lung and spleen have

distinctly different levels of expression for the two types of

Ca2 + ATPases.

Fig. 2. Western blotting of SPCA1 in a variety of tissue membranes. Tissue

membranes (15–30 Ag) and TM4 cell homogenate were resolved on a 7.5%

SDS-PAGE gel and the proteins were transferred onto nitrocellulose. The

nitrocellulose was incubated with anti-SPCA1 antibody diluted 1 in 100 for

1.5 h at room temperature and with secondary anti-rabbit IgG conjugated to

horseradish peroxidase for 1 h at room temperature. The blot was then

incubated with SuperSignal West Pico substrate (Pierce) according to the

manufacturer’s instructions and the blot was used to expose Kodak BioMax

MR film, which was developed using an XO-graph machine. The lanes

represent the following: (1) rat brain (15 Ag), (2) rat lung (30 Ag), (3) rat liver(30 Ag), (4) rat testis (30 Ag), (5) rat heart (30 Ag), (6) rat fat pads (30 Ag), (7)pig aorta (30 Ag), (8) rat kidney (30 Ag), (9) rat spleen (30 Ag) and (10) mouse

TM4 cells.

As SPCA is believed to localised to the Golgi apparatus

within cells, the amount of Golgi present in the tissue

membranes was assessed by measuring the activities of

two Golgi-associated enzymes (UDPase and a-mannosi-

Fig. 3. Western blotting of SERCA in a variety of tissue membranes. Tissue

membranes (30 Ag) and TM4 cells were resolved on a 7.5% SDS-PAGE

before being transferred onto nitrocellulose. The nitrocellulose was

incubated with anti-SERCA antibody diluted 1 in 500 for 1.5 h at room

temperature and with secondary anti-mouse IgG conjugated to horseradish

peroxidase, for 1 h at room temperature, according to the supplier’s

instructions. The blot was incubated with SuperSignal West Pico (Pierce)

according to the manufacturer’s instructions and was used to expose Kodak

BioMax MR film, which was developed using an XO-graph machine. The

lanes represent the following: (1) rat kidney, (2) rat lung, (3) rat brain, (4)

pig aorta, (5) mouse TM4 cells, (6) rat fat pads, (7) rat heart, (8) rat testis

and (9) rat liver and (10) rat spleen (all microsomal membranes 30 Ag).

Table 2

Ca2 + pump activity in the absence and presence of 2 AM thapsigargin

Tissue Initial rate of

Ca2 + uptake

(nmol/min/mg)

Initial rate of

Ca2 + uptake in

the presence

of 2 AMthapsigargin

(nmol/min/mg)

Initial rate of

Ca2 + uptake

due to SERCA

(nmol/min/mg)

Ratio of

TGInsensitive

activity/total

activity

‘Total activity’ ‘TGInsensitive

activity’

‘Total�TGInsensitive’

Testis 0.740F 0.181 0.250F 0.005 0.49F 0.19 0.34

Heart 8.830F 0.060 0.260F 0.017 8.57F 0.08 0.03

Fat Pads 0.390F 0.068 0.320F 0.034 0.07F 0.1 0.82

Spleen 0.026F 0.006 0.021F 0.004 0.005F 0.01 0.81

Liver 0.600F 0.070 0.023F 0.003 0.577F 0.07 0.04

Kidney 0.110F 0.015 0.055F 0.009 0.055F 0.02 0.50

Brain 3.085F .0513 1.420F 0.090 1.67F 0.06 0.46

Lung 0.430F 0.101 0.067F 0.005 0.36F 0.10 0.16

Aorta 1.480F 0.204 0.815F 0.025 0.67F 0.22 0.55

These values are presented as the meanF S.E. of at least three or more

determinations. TGInsensitive stands for thapsigargin-insensitive Ca2 + uptake

activity. All tissue membranes were derived from rat except aorta which

was derived from pig.


dase), the activities of which are shown in Table 1. The

activity of UDPase in the different membranes is quite

variable, with the activity being particularly high in liver

and kidney (which showed little expression of SPCA1). The

activity of a-mannosidase was again variable between the

different tissue membranes, with the highest activity occur-

ring in epididymal fat pads, liver and heart. These results

therefore showed little correlation between the amount of

SPCA1 and Golgi apparatus present within these tissue

membranes.

In order to measure the activity of SPCA in the different

tissue membranes, rates of ATP-driven Ca2 +-uptake were

monitored. To minimise the contribution of other known

Ca2 +-ATPases and Ca2 +-uptake processes which might be

present within these membranes, the assays were undertaken

in the presence of 2 AM vanadate and 2 mM sodium azide to

inhibit plasma membrane Ca2 +-ATPase activity [48] and

mitochondrial Ca2 +-uptake, respectively. In addition, these

activities were measured in the presence and absence of 2

AM thapsigargin (which inhibits SERCA) to determine the

contributions of SPCA and SERCA activities within these

tissue membranes. It has been previously shown that the

Golgi Ca2 + ATPase is unaffected by both 2 AM thapsigargin

and 2 AM vanadate [9,45]. As the plasma membrane Ca2 +-

ATPase (PMCA) is extremely sensitive to vanadate (i.e.

complete inhibition occurs at about 2 AM [48]) this was

added to the assay buffer. However, since SERCA is also

inhibited by vanadate (albeit at much higher concentrations

[49]), we also wanted to make sure that our ‘total activity’

was not substantially affected by the 2 AM vanadate present

in the buffer causing SERCA inhibition. Therefore, in order

to assess this possibility, the Ca2 + pump uptake activity was

measured in a number of tissues membranes which express

the main SERCA isoforms: i.e. heart for SERCA 2a iso-

form, brain for SERCA2b and testis for SERCA2b and

SERCA3 [50,51]. The degree of inhibition in the presence

of 2 AM vanadate compared to its absence was not greatly

affected in all three tissue membranes tested (i.e. heart, brain

and testis membranes showed only 3%, 0% and 12%

inhibition, respectively).

Table 1

Activity of UDPase and a-mannosidase in tissue membranes

Tissue UDPase activity

(nmol Pi/min/mg)

a-Mannosidase II activity

(nmol pNP/min/mg)

Testis 300F 1 3.45F 0.14

Heart 105F 1 7.51F 0.04

Fat pads 357F 2 8.44F 0.09

Spleen 128F 1 5.41F 0.12

Liver 1440F 30 5.39F 0.44

Kidney 780F 4 7.62F 0.14

Brain 90F 1 6.52F 1.03

Lung 155F 1 2.20F .0.25

Aorta 78F 2 0.83F 0.04

These values presented are the meanF S.E. of at least three or more

determinations. All tissues are derived from rat except aorta which was

derived from pig.

Table 2 lists the initial rates of Ca2 + uptake for the

different rat tissue membranes, in the presence and absence

of 2 AM thapsigargin. When measured in the presence of

thapsigargin (i.e. to monitor the thapsigargin-insensitive

activity presumably due to SPCA), brain and porcine aorta

had by far the highest levels, followed by epididymal fat

pads, heart and testis. The other tissues tested had relatively

very little SPCA activity. Surprisingly, testis membranes had

relatively low levels of activity compared to brain consid-

ering the relative abundance of SPCA protein from the

immunoblots. ‘Total’ Ca2 +-ATPase activity (i.e. activities

in the absence of thapsigargin, where SERCA would likely

have a substantial contribution) or total activity minus the

thapsigargin-insensitive activity (which would correspond

mainly with the SERCA activity) was highest in heart,

brain, porcine aorta and testis, as expected from the SERCA

immunoblots results. When comparing the activity of SPCA

within tissues that had reasonable amounts of total Ca2 +

pump activity, it was found that brain, aorta, testis and fat

pads, all had considerable levels of thapsigargin-insensitive

activity corresponding to 34% or more of the total Ca2 +

pump activity.

In order to investigate the subcellular localisation of

SPCA and compare it to the location of the Golgi complex

and SERCA, TM4 cells and HuASMC (contractile pheno-

type) were used, since they expressed SPCA and SERCA to

reasonable levels. These cells were therefore stained with

the anti-SERCA antibody (Y1F4), anti-SPCA1 antibody

and with WGATR, which binds to and stains the Golgi

complex. Fig. 4 shows the results of immunofluorescence

staining of TM4 and HuASMC. TM4 cells stained with the

anti-SPCA antibody revealed labelling around the nucleus,

consistent with localisation in the Golgi, as staining with

WGATR of the same cell revealed a very similar distribu-

Fig. 4. Immunofluorescence staining of TM4 cells and human primary

aortic smooth muscle cells. TM4 cells were cultured on scratched glass

coverslips; primary human aortic smooth muscle cells were cultured on

gelatine-coated glass cover slips. The cells were washed with PBS and fixed

with 4% paraformaldehyde in PBS and 2% formaldehyde in PBS, before

being permeabilised for 5 min at room temperature with 0.1% Triton X-100

in PBS. The cells were incubated with either anti-SPCA1 diluted 1 in 100 in

PBS or anti-SERCA antibody diluted 1 in 100 for 45 min. The cells were

washed with PBS before being incubated with the appropriate secondary

FITC-conjugated IgG according to the manufacturer’s instructions. As an

alternative to antibody, the cells were incubated for 45 min at room

temperature with WGATR diluted to a concentration of 2 Ag/ml in PBS and

mounted on glass slides using Hydramount and visualised as for the

immunostained cells. The bar in each of the TM4 cell diagrams represents

50 Am. The bar in each of the human primary aortic smooth muscle cell

diagrams represents 100 Am. (A) TM4 cell stained with anti-SPCA1

antibody (1/100 dilution) and anti-rabbit IgG FITC (1/64 dilution). (B)

TM4 cell stained with WGATR (2 Ag/ml in PBS). (C) TM4 cell stained

with Y1F4 anti-SERCA antibody (1/100 dilution) and anti-mouse IgG

FITC (1/40 dilution). (D) Primary human aortic smooth muscle cell

(HuASMC) stained with anti-Pmr1 (1/100 dilution) and anti7-rabbit IgG

FITC (1/64 dilution). (E) HuASMC stained with WGATR (2 Ag/ml in

PBS). (F) HuASMC stained with Y1F4 anti-SERCA monoclonal antibody

(1/100 dilution) and anti-mouse IgG FITC (1/64 dilution).


tion. Such staining is also consistent with previous immu-

nostaining of human SPCA in keratinocytes [17]. Staining

of TM4 cells with the Y1F4 anti-SERCA antibody revealed

a more diffuse staining pattern of the majority of the cell,

with the exception of the nucleus, consistent with local-

isation throughout the ER. HuASMCs co-labelled with the

anti-SPCA antibody and WGATR also stained a distinct

area around the nucleus. There was no significant staining

observed with either the secondary antibody alone or with

pre-immune serum and FITC secondary antibody.

5. Discussion

From RT-PCR of rat tissue samples and from Western

blotting of rat tissue membranes, it has been shown that the

SPCA1 protein is highly expressed in both brain and testis.

Analysis of rat poly (A+) RNA from several tissues by

Northern blotting by Gunteski-Hamblin et al. [16] also

showed that the levels of message for SPCA for brain and

testis were higher relative to other tissues. Hu et al. [19] also

analysed the levels of poly (A+) RNA from several human

tissues by Northern blotting, showing that the level of

message for ATP2C1a and ATP2C1b for brain was lower

compared with heart, skeletal muscle and kidney, inconsis-

tent with our findings in rat tissues.

The secretory function of both the brain and testis is

important. The brain in particular secretes many neuro-

transmitters from vesicles. It has been shown that SPCA is

important for the transportation of Ca2 + into secretory

vesicles in neuroendocrine cells [36] and that Ca2 + is

important for the regulation of Golgi transport [37], thus

SPCA may be abundant in the brain as it may regulate the

secretion of neurotransmitters and secretory proteins.

Many of the testicular cell types have an important

secretory role in paracrine signalling; Leydig cells are

known to secrete testosterone [38], myoid cells are able to

secrete adenosine [39] and Sertoli cells secrete a variety of

hormones [40] and other factors to aid the developing

sperm. As many testicular cell types have important secre-

tory function, it may be expected that the level of SPCA1

expression may be higher in testis in order to aid the

synthesis of secretory proteins.

The highest SPCA activity was observed in brain, the

expression of SPCA in testis was at a comparable level to

that of brain, yet the activities were much lower in this

tissue. The reason for this is unclear, however, one possi-

bility is that SPCA in testis is regulated by a modulatory

protein, which decreases its activity, a suggestion previously

made about human SPCA1d in keratinocytes to explain a

reduction in Vmax in Ca2 + transport [17]. There also appears

to be some evidence for the role of SPCA2 in inhibiting the

activity of SPCA1 and SERCA, in tissues where both are

expressed, while SPCA2 alone appears to have little or no

activity (Jo Vanoevelen and Ludwig Missiaen, personal

communication).

Furthermore, as the proportion of SPCA activity com-

pared to total Ca2 + ATPase activity in brain, aorta, testis and

epididymal fat pads was particularly high, this would

indicate that SPCA may play a major role in the refilling

of a distinct and abundant type of Ca2 + store within these

tissues.


Immunofluorescence staining of both the TM4 cells and

human primary aortic smooth muscle cells has revealed that

SPCA1 is localised at or near the Golgi in these cell types.

Potentially SPCA may play a fundamental role in fertility as

the function of the Sertoli cell is to secrete factors, such as

purines [39], growth factors [40] and plasminogen activator

[41], and to respond to paracrine signalling in order to

nurture immature sperm. Interestingly it has been shown

that patients suffering from Klinefelter’s syndrome, a con-

dition causing male infertility, have dysfunctional secretory

mechanisms within their Sertoli cells [42].

In aortic cells (where our results would indicated that

around 50% of the Ca2 + stores are loaded by SPCA), SPCA

could act as a backup to SERCA Ca2 + pumps in controlling

the contractile status and therefore regulating vasodilatation/

vasoconstriction.

To summarize, in both TM4 cells and aortic smooth

muscle cells, SPCA may be important for the filling of an

agonist-releasable store of Ca2 +, a role suggested for SPCA

in A7r5 cells [21], HeLa cells [14] and keratinocytes [43]. In

these cells the generation of baseline Ca2 + spiking, which

may be important in modulating spatio-temporal Ca2 +

patterns and oscillations, has been attributed to SPCA.

Acknowledgements

We would like to thank the British Heart Foundation for

a PhD studentship to L.L.W. and a project grant to M.W

(BHF grant number PG/03/082/15735). Marukh Akhtar is

thanked for preliminary experiments. We would also like to

thank Dr. J.M. East from the University of Southampton,

UK for the gift of Y1F4 anti-SERCA antibody. Frances

Turner and Dr. N. Hotchin (School of Biosciences,

University of Birmingham) are also thanked for advice on

immunofluorescence and use of the fluorescence micro-

scope. Prof. Ludwig Missiaen is also thanked for helpful

discussions.

References

[1] M.J. Berridge, Inositol trisphosphate and calcium signalling, Nature

361 (1993) 315–325.

[2] M.J. Berridge, M.D. Bootman, P. Lipp, Calcium—a life and death

signal, Nature 395 (1998) 645–648.

[3] T. Chakraborti, S. Das, M. Mondal, S. Roychoudhury, S. Chakraborti,

Oxidant, mitochondria and calcium: an overview, Cell. Signal. 11

(1999) 77–85.

[4] E. Carafoli, M. Brini, Calcium pumps: structural basis for and mech-

anism of calcium transmembrane transport, Curr. Opin. Chem. Biol. 4

(2000) 152–161.

[5] F. Wuytack, L. Raeymaekers, L. Missiaen, Molecular physiology

of the SERCA and SPCA pumps, Cell Calcium 32 (2002) 279–

305.

[6] R.A. Smith, M.J. Duncan, D.T. Moir, Heterologous protein secretion

from yeast, Science 229 (1985) 1219–1224.

[7] R. Serrano, M.C. Kielland-Brandt, G.R. Fink, Yeast plasma membrane

ATPase is essential for growth and has homology with (Na+ K+) K+-

and Ca2 +-ATPases, Nature 319 (1986) 689–693.

[8] H.K. Rudolph, A. Antebi, G.R. Fink, C.M. Buckley, T.E. Dorman, J.

LeVitre, L.S. Davidow, J.I. Mao, D.T. Moir, The yeast secretory path-

way is perturbed by mutations in PMR1, a member of a Ca2 + ATPase

family, Cell 58 (1989) 133–145.

[9] A. Sorin, G. Rosas, R. Rao, PMR1, a Ca2 +-ATPase in yeast Golgi,

has properties distinct from sarco/endoplasmic reticulum and plasma

membrane calcium pumps, J. Biol. Chem. 272 (1997) 9895–9901.

[10] A. Antebi, G.R. Fink, The yeast Ca2 +-ATPase homologue, PMR1, is

required for normal Golgi function and localizes in a novel Golgi-like

distribution, Mol. Biol. Cell 3 (1992) 633–654.

[11] V.K. Ton, D. Mandal, C. Vahadji, R. Rao, Functional expression in

yeast of the human secretory pathway Ca2 +, Mn2 +-ATPase defective

in Hailey–Hailey disease, J. Biol. Chem. 277 (2002) 6422–6427.

[12] G. Durr, J. Strayle, R. Plemper, S. Elbs, S.K. Klee, P. Catty, D.H.

Wolf, H.K. Rudolph, The medial-Golgi ion pump Pmr1 supplies the

yeast secretory pathway with Ca2 + and Mn2 + required for glyco-

sylation, sorting and endoplasmic reticulum-associated protein deg-

radation, Mol. Biol. Cell 9 (1998) 1149–1162.

[13] P. Pinton, T. Pozzan, R. Rizzuto, The Golgi apparatus is a inositol

1,4,5-trisphosphate-sensitive Ca2 + store, with functional properties

distinct from those of the endoplasmic reticulum, EMBO J. 17

(1998) 5298–5308.

[14] K. Van Baelen, J. Vanoevelen, G. Callewaert, J.B. Parys, H. De Smedt,

L. Raeymaekers, R. Rizzuto, L. Missiaen, F. Wuytack, The contribu-

tion of the SPCA1 Ca2 + pump to the Ca2 + accumulation in the Golgi

apparatus of HeLa cells assessed via RNA-mediated interference, Bio-

chem. Biophys. Res. Commun. 306 (2003) 430–436.

[15] L. Missiaen, J. Vanoevelen, K. Van Acker, L. Raeymaekers, J.B. Parys,

G. Callewaert, F. Wuytack, H. De Smedt, Ca2 + signals in Pmr1-GFP-

expressing COS-1 cells with functional endoplasmic reticulum, Bio-

chem. Biophys. Res. Commun. 294 (2002) 249–253.

[16] A. Gunteski-Hamblin, D.M. Clarke, G.E. Shull, Molecular cloning

and tissue distribution of alternatively spliced mRNAs encoding pos-

sible mammalian homologues of the yeast secretory pathway calcium

pump, Biochemistry 31 (1992) 7600–7608.

[17] R.J. Fairclough, L. Dode, J. Vanoevelen, J.P. Andersen, L. Missiaen,

L. Raeymaekers, F. Wuytack, A. Hovnanian, Effect of Hailey–Hailey

disease mutations on the function of a new variant of human secretory

pathway Ca2 +/Mn2 +-ATPase (hSPCA1), J. Biol. Chem. 278 (2003)

24721–24730.

[18] R. Sudbrak, J. Brown, C. Dobson-Stone, S. Carter, J. Ramser, J.

White, E. Healy, M. Dissanayake, M. Larregue, M. Perrussel, H.

Lehrach, C.S. Munro, T. Strachan, S. Burge, A. Hovnanian, A.P.

Monaco, Hailey –Hailey disease is caused by mutations in

ATP2C1 encoding a novel Ca2 + pump, Hum. Mol. Genet. 9 (2000)

1131–1140.

[19] Z. Hu, J.M. Bonifas, J. Beech, G. Bench, T. Shigihara, H. Ogawa, S.

Ikeda, T. Mauro, E.H. Epstein, Mutations in ATP2C1, encoding a

calcium pump, cause Hailey–Hailey disease, Nat. Genet. 24 (2000)

61–65.

[20] S.C. Tovey, R.G. Godfrey, P.J. Hughes, M. Mezna, S.D. Minchin, K.

Mikoshiba, F. Michelangeli, Identification and characterization of

inositol 1,4,5-trisphosphate receptors in rat testis, Cell Calcium 21

(1997) 311–319.

[21] L. Missiaen, J. Vanoevelen, J.B. Parys, L. Raeymaekers, H. De Smedt,

G. Callewaert, C. Erneux, F. Wuytack, Ca2 + uptake and release prop-

erties of a thapsigargin-insensitive nonmitochondrial Ca2 + store in

A7r5 and 16HBE14o-cells, J. Biol. Chem. 277 (2002) 6898–6902.

[22] U.K. Laemmli, Cleavage of structural proteins during the assembly of

the head of bacteriophage T4, Nature 227 (1970) 680–685.

[23] J.M. Gershoni, F.E. Davis, G.E. Palade, Protein blotting in uniform or

gradient electric fields, Anal. Biochem. 144 (1985) 32–40.

[24] N.J. Kuhn, A. White, The role of nucleoside diphosphatase in a

uridine nucleotide cycle associated with lactose synthesis in rat mam-

mary-gland Golgi apparatus, Biochem. J. 168 (1977) 423–433.


[25] E. Brandan, B. Fleischer, Orientation and role of nucleosidediphos-

phatase and 5V-nucleotidase in Golgi vesicles from rat liver, Bio-

chemistry 21 (1982) 4640–4645.

[26] D.J. Morre, D.M. Morre, H.G. Heidrich, Subfractionation of rat liver

Golgi apparatus by free-flow electrophoresis, Eur. J. Cell Biol. 31

(1983) 263–274.

[27] Y.T. Li, Studies on the glycosidases in jack bean meal: I. Isolation

and properties of alpha-mannosidase, J. Biol. Chem. 242 (1967)

5474–5480.

[28] K.W. Moremen, Isolation of a rat liver Golgi mannosidase II clone by

mixed oligonucleotide-primed amplification of cDNA, Proc. Natl.

Acad. Sci. U. S. A. 86 (1989) 5276–5280.

[29] K.W. Moremen, O. Touster, P.W. Robbins, Novel purification of the

catalytic domain of Golgi alpha-mannosidase II. Characterization and

comparison with the intact enzyme, J. Biol. Chem. 266 (1991)

16876–16885.

[30] C.L. Longland, M. Mezna, U. Langel, M. Hallbrink, U. Soomets, M.

Wheatley, F. Michelangeli, J. Howl, Biochemical mechanisms of cal-

cium mobilisation induced by mastoparan and chimeric hormone-

mastoparan constructs, Cell Calcium 24 (1998) 27–34.

[31] F. Michelangeli, Measuring calcium uptake and inositol 1,4,5-triphos-

phate-induced calcium release in cerebellar microsomes using Fluo-3,

J. Fluoresc. 1 (1991) 203–206.

[32] F. Michelangeli, The effects of amino acid-reactive reagents on the

functioning of the inositol 1,4,5-trisphosphate-sensitive calcium chan-

nel from rat cerebellum, Cell. Signal. 5 (1993) 33–39.

[33] M. Mezna, F. Michelangeli, Alkali metal ion dependence of inositol

1,4,5-trisphosphate-induced calcium release from rat cerebellar micro-

somes, J. Biol. Chem. 270 (1995) 28097–28102.

[34] C. Schubert Wright, Structural comparison of the two distinct sugar

binding sites in wheat germ agglutinin isolectin II, J. Mol. Biol. 178

(1984) 91–104.

[35] R.E. Tunwell, J.W. Conlan, I. Matthews, J.M. East, A.G. Lee, Defi-

nition of surface-exposed epitopes on the (Ca(2+)-Mg2+)-ATPase of

sarcoplasmic reticulum, Biochem. J. 279 (1991) 203–212.

[36] K.J. Mitchell, P. Pinton, A. Varadi, C. Tacchetti, E.K. Ainscow, T.

Pozzan, R. Rizzuto, G.A. Rutter, Dense core secretory vesicles

revealed as a dynamic Ca(2+) store in neuroendocrine cells with

a vesicle-associated membrane protein aequorin chimaera, J. Cell

Biol. 155 (2001) 41–51.

[37] A. Porat, Z. Elazar, Regulation of intra-Golgi membrane transport by

calcium, J. Biol. Chem. 275 (2000) 29233–29237.

[38] H. Ostrer, Sexual differentiation, Semin. Reprod. Med. 18 (2000)

41–49.

[39] D.P. Gelain, L.F. de Souza, E.A. Bernard, Extracellular purines from

cells of seminiferous tubules, Mol. Cell. Biochem. 245 (2003) 1–9.

[40] T. Meehan, S. Schlatt, M.K. O’Bryan, D. deKrester, K.L. Loveland,

Regulation of germ cell and Sertoli cell development by activin,

follistatin, and FSH, Dev. Biol. 220 (2000) 225–237.

[41] Y.X. Liu, K. Liu, H.M. Zhou, Q. Du, Z.Y. Hu, R.J. Zou, Hormonal

regulation of tissue-type plasminogen activator and plasminogen ac-

tivator inhibitor type-1 in cultured monkey Sertoli cells, Hum.

Reprod. 10 (1995) 719–727.

[42] Y. Yamamoto, N. Sofikitis, Y. Mio, D. Loutradis, A. Kaponis, I.

Miyagawa, Morphometric and cytogenetic characteristics of testic-

ular germ cells and Sertoli cell secretory function in men with

non-mosaic Klinefelter’s syndrome, Hum. Reprod. 17 (2002)

886–896.

[43] G. Callewaert, J.B. Parys, H. De Schmedt, L. Raeymaekers, F.

Wuytack, J. Vanoevelen, K. Van Baelen, A. Simoni, R. Rizzuto, L.

Missiaen, Similar Ca(2+)-signaling properties in keratinocytes and in

COS-1 cells overexpressing the secretory-pathway Ca(2+)-ATPase

SPCA1, Cell Calcium 34 (2003) 157–162.

[44] G.E. Rainger, P. Stone, C.M. Morland, G.B. Nash, A novel system for

investigating the ability of smooth muscle cells and fibroblasts to

regulate adhesion of flowing leukocytes to endothelial cells, J. Immu-

nol. Methods 255 (2001) 73–88.

[45] L. Missiaen, K. VanAcker, J.B. Parys, H. DeSmedt, K, VanBaelen,

A.F. Weidema, J. Vanevelen, L. Raeymaekers, J. Renders, G. Call-

ewaert, R. Rizzuto, F. Wuytack, Baseline cytosolic Ca2 + oscillations

derived from a non-endoplasmic reticulum Ca2 + store, J. Biol. Chem.

276 (2001) 39161–39170.

[46] S.A. Rudge, P.J. Hughes, G.R. Brown, C.J. Kirk, Inositol lipid-me-

diated signalling in response to endothelin and ATP in the mammalian

testis, Mol. Cell. Biochem. 149 (1995) 161–174.

[47] J. Howl, R.A. Rudge, R.A. Lavis, R.H. Michell, C.J. Kirk, M. Wheat-

ley, Rat testicular myoid cells express vasopressin receptors: receptor

structure signal transduction and developmental regulation, Endocri-

nology 136 (1995) 2206–2213.

[48] E. Carafoli, M. Zurini, The Ca2 +-pumping ATPase of Plasma mem-

branes: purification, reconstitution and properties, Biochim. Biophys.

Acta 683 (1982) 279–301.

[49] J. Lytton, M. Westlin, S.E. Burk, G.E. Shull, D.H. MacLennan, Func-

tional comparisons between isoforms of the sarcoplasmic or endoplas-

mic reticulum family of calcium pumps, J. Biol. Chem. 267 (1992)

14483–14489.

[50] K.D. Wu, W.S. Lee, J. Wey, D. Bungard, J. Lytton, Localization and

quantification of endoplasmic reticulum Ca(2+)-ATPase isoform tran-

scripts, Am. J. Physiol. 269 (1995) C775–784.

[51] P.J. Hughes, H. McLellan, D.A. Lowes, S.Z. Khan, J.G. Bilmen, S.C.

Tovey, R.E. Godfrey, R.H. Michell, C.J. Kirk, F. Michelangeli, Es-

trogenic alkylphenols induce cell death by inhibiting testis endoplas-

mic reticulum Ca2 + pumps, Biochem. Biophys. Res. Commun. 277

(2000) 568–574.

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